BRB 747A, 2109 Adelbert Road, Cleveland, Ohio 44106
Dye-terminator sequencing is performed using a modified protocol supplied by Applied Biosystems BigDye Terminator 1.1 and 3.1 Cycle sequencing kits. All reactions are analyzed using the 3730 Genetic Analyzer. Sequencing can be performed on a variety of templates including PCR product, plasmids, and BACs.
Samples are processed on Monday and Thursday mornings only (We no longer process samples on a daily basis). Data will be ready the following morning. Samples and primers must be submitted in separate tubes. Please make sure to clearly label each tube with the appropriate name, the date, and your name or initials. Large sets of sequencing can be submitted in a 96-well plate format with a volume of 10ul per reaction. There is a price reduction for processing more than 96 reactions at a time. For just $1 per sample, the Core provides an Exo-SAP treatment service to PCR products if no other clean-up step has been preformed. Please provide at least 10ul of PCR product if you would like this service. Finally, fill out the Sequencing Request form and bring your samples to the Core on the 7th floor of the BRB.
|Type of Template:||Quantity:|
|Plasmid||100-300ng per reaction|
|Short PCR product (<500bp)||50-100ng per reaction|
|Long PCR product (>500bp)||100-200ng per reaction|
|Primer||2ul of 20uM concentration per reaction|
All primers must be submitted in separate tubes at a 20uM concentration, clearly labeled with the date and your name or initials. Please provide at least 2ul of primer per reaction and list an annealing temperature on the request form. The Genomics Core does offer universal primers at no cost to the user.
Universal primers stocked by the Core
|BGH reverse:||5’ TAGAAGGCACAGTCGAGG|
|M13 Forward:||5’ GTTTTCCCAGTCACGAC|
|M13 Reverse||5’ AACAGCTATGACCATG|
|T7 promoter||5’ TAATACGACTCACTATAGGG|
Data can be accessed through the Helix fileserver which is accessible to all systems on the Case network. An account for your lab can be created where data files can be securely stored and retrieved. Please email the Genetics Department Computer Systems Administrator, Ulrich Neuss (Uli) at firstname.lastname@example.org to set up an account. In the e-mail please specify your name, e-mail address, your principal investigator, and explain that you would like access to the facility to get your data.